《Chapter13 Enzyme applications and research methods.ppt》由会员分享,可在线阅读,更多相关《Chapter13 Enzyme applications and research methods.ppt(15页珍藏版)》请在得力文库 - 分享文档赚钱的网站上搜索。
1、Chapter13EnzymeapplicationsandResearchmethods,Outline,AssayofenzymeactivitiesMethodsforexpressingenzymeactivitiesMethodsfordeterminingenzymeactivitiesSeparationandpurificationofenzymes(canbeskipped)Enzymeengineering(self-study),Enzymeactivity,Enzymeactivity=molesofsubstrateconvertedperunittime=rater
2、eactionvolume.Enzymeactivityisameasureofthequantityofactiveenzymepresentandisthusdependentonconditions,whichshouldbespecified.TheSI(TheInternationalSystem)unitisthekatal,1katal=1mols1,butthisisanexcessivelylargeunit.Amorepracticalandcommonlyusedvalueis1enzymeunit(U)=1molmin1.1U=16.67nanokatalsand1ka
3、t=6107U,Specificactivity,Thespecificactivityofanenzymeistheactivityofanenzymepermilligramoftotalprotein(expressedinmolmin1mg1).Specificactivitygivesameasurementoftheactivityoftheenzyme.Itistheamountofproductformedbyanenzymeinagivenamountoftimeundergivenconditionspermilligramoftotalprotein.Specificac
4、tivityisequaltotherateofreactionmultipliedbythevolumeofreactiondividedbythemassoftotalprotein.TheSIunitiskatalkg1,butamorepracticalunitismolmg1min1.,Enzymeactivityassay,AllenzymeassaysmeasureeithertheconsumptionofsubstrateorproductionofproductovertimeEnzymeassayconditionsAlargeexcessofthesubstrateIn
5、itialvelocityOptimalreactionconditions(optimalpH,optimaltemperature,optimalionicstrength,etc).AssaymethodsDirectassayIndirectassayCoupledassay,Lightabsorption,Lightabsorption,Directassayofcytochromecoxidase,IndirectassayofDihydrooratedehydrogenase,Lightabsorption,CoupledassayofHexosekinase,Enzymepur
6、ificationisextractionofasingleenzyme/proteinfrome.g.cells,tissue,etc.whichmaycontainmorethan1000differentproteinsandlotsofotherbiomoleculesKeepstructureandactivityintactEachenzymerequiresaspecificstrategyforpurificationHowispurificationmeasured?,Enzymepurification,SelectthesourcesEstablishtheessayme
7、thodDesignthestrategyPreparingextractsforpurificationDisruptionandhomogenizationofcells,tissue,etc.ClearingofextractsChoosepurificationmethodsDeterminepurity,PurificationSteps,PrecipitationmethodsSeparationbasedonmolecularsizeSeparationbasedonchargeSeparationbasedonspecificinteractionwithotherbiomol
8、eculesSeparationbasedonotherprinciplesExamples:AmmoniumsulfateprecipitationGelfiltrationIon-exchangechromatographyBio-affinitychromatographyHydrophobicinteractionchromatography;hydroxyapatitechromatography,Maintypesofpurificationmethods,DeterminationofspecificactivityPurificationtablePhysicalmethods
9、:SDS-PAGEGelfiltration,Howispurificationmeasured?,Purificationtable,Measureforeachpurificationstep:Thevolumeoftheenzymesolution(ml)Theproteincontentofthesolution(mg.ml-1)Theactivityoftheenzymesolution(U.ml-1)Totalamountofenzyme(U):Activity(U.ml-1)xvolume(ml)Specificactivity(U.mg-1):Activity(U.mg-1)/
10、proteincontent(mg.ml-1)Yield(%):Totalamountofenzymeafterapurificationstep/totalamountofenzymebeforethatstepPurificationfactor:Specificactivityofenzymeafterapurificationstep/specificactivitybeforethatstep,Purificationtable(example),ImmobilizedenzymesArtificialenzymesSite-directedmutagenesisofenzymesHybridenzymesAbzymes,Enzymeengineering,